140 Albert L. Lehninger, et al. 



preparations of mitochondria (Pullman and Racker, 1956; 

 Polls and Shmukler, 1957; Stern and Timonen, 1955; Beyer 

 and Glomset, 1956) and in fly mitochondria (Sacktor, 1954). 



We have recently separated a mitochondrial factor having 

 some properties in common with mitochrome and the iso- 

 octane-extractable factor of Hiilsmann and co-workers, but 

 which has other rather striking effects on electron transport 

 and coupled phosphorylation which have not yet been des- 

 cribed and which may be important in intramitochondrial 

 control of respiration (Remmert and Lehninger, 1959). The 

 factor to be described may contain a more native form of 

 mitochrome or its 250octane-soluble activity described above; 

 but it is probable that it is a mixture of factors affecting 

 energy coupling. 



Freshly prepared rat liver mitochondria are subjected to 

 9 kcyc. sonic oscillation and the debris is extracted with 

 dilute sucrose-KCl solutions. The extract is clarified by 

 centrifugation at 115,000 g for 30 minutes and then dialysed 

 exhaustively. Protein precipitating from this extract when the 

 pH is adjusted to 5 • 5 represents the basic starting material 

 for our tests. 



When tested in relatively high concentrations, this soluble 

 mitochondrial protein fraction (called R factor) causes 

 uncoupling of phosphorylation from oxidation of p-hydroxy- 

 butyrate, inhibition of the ATP-^^p. exchange reaction, and 

 stimulation of ATPase activity, as is shown by the data in 

 Table III. The preparation of R factor itself shows no activity 

 in oxidative phosphorylation or in the ATP-^^p. exchange and 

 only negligible ATPase activity. These properties of the R 

 factor preparation, taken at face value, suggest it is a naturally 

 occurring uncoupling agent present in fresh mitochondria 

 (but presumably in "masked" form) which is released on 

 sonication. In the reactions shown in Table I it behaves 

 like DNP and gives the same effects as preparations of mito- 

 chrome or its i^ooctane-soluble component. However, further 

 study shows it to be very different from DNP and possibly 

 different from the materials described by others. 



